Recently, some new cell culture technologies, including special scaffolds (eg: gel, fiber) to form three-dimensional (3-D) spherical aggregates, have been developed. In case of hepatocyte, these aggregates have been observed to maintain higher level of hepatocytes specific functions including cytochrome P450 activity and albumin secretion. In addition, in the cell-based assays for drug discovery, high throughput screening (HTS) is demanded. Consequently, we studied 3-D culture methods using micro structural scaffolds that are divided by micro-sized wall on the surface, which provide simple handling to be applicable for HTS. Our attempt was to build spheroids with diameter ranging from 50 to 200μm because the size is regarded as suitable for nutrition supply into the center of the spheroid. We prepared cell culture plates, made of polystyrene, with micro-space arrayed regularly on bottom surface (Figure).
The shape of each micro-space is a square with 200μm width and 50μm or 100μm depth (Micro-Space Culture Plate). Primary hepatocytes were cultured on 24-well Micro-Space Culture Plate for 5-6 days by the same method as the monolayer culture on flat plates (Initial cell density: 0.2-0.4 million in 500μl). Medium change was done after 4 hr from culture initiation and everyday thereafter. During initial 4 hr period, cells attached to the surface in each micro-space, which is similar to the case on flat plate. The cells began to aggregate and formed spheroid like structure in the center of some micro-spaces at day 3 of culture. This structure was presented in almost all micro-spaces at day 5. We analyzed hepatocyte specific functions at day 3 and 5. mRNA expression of major cytochrome P450 enzymes, CYP1A2, 3A4 and 2C9, were higher than those of monolayer culture by 3-5 fold, 3-5 fold and 2-3 fold, respectively . Also, mRNA expression of phase II enzymes, UGTs, was highly maintained compared to those with conventional culture method. Expression of UGT1A1 was higher than that of monolayer culture by 2-5 fold. These observations indicated that Micro-Space Culture Plate could be employed for 3-D cell culture by the same handling as used for flat plate. Furthermore, In case of hepatocytes, fundamental functions could be maintained during the culture period so that the plate is expected to be useful for an HTS screening tool to evaluate drug metabolism and toxicity.